Figure 3

Rac1 inhibition abrogates IR-induced G ₂ /M checkpoint activation. (A) MCF-7 cells were incubated for 1 hour in the presence or absence of 100 μM NSC23766, treated with/without 20-Gy IR, and incubated for an additional 2 hours at 37°C. Cdc2 was immunoprecipitated from cell lysates and analyzed for Cdc2-Tyr15 phosphorylation by immunoblotting (Cdc2-Tyr15) and Cdc2 activity by kinase assay (Cdc2 activity). Amount of Cdc2 protein in the immunoprecipitates was assessed by immunoblotting (Cdc2 IP-WB). (B) Cells were treated as described and analyzed for mitotic cells with fluorescence-activated cell sorting (FACS), which contains both 4N-DNA content and Histone H3-Ser10 phosphorylation, as described in Materials and methods. Upper panel: the histograms shown are representative FACS analyses for mitotic cells in samples treated with/without IR in the presence or absence of NSC23766. The location of mitotic cells in each sample is indicated (M). Lower panel: the bar graph depicts the percentage of mitotic cells and is shown as mean ± SD of triplicate samples. * P = < 0.001 (n = 3), significant difference from cells exposed to IR in the absence of NSC23766.